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Imunologia e Infecção

En Ex vivo analyse for å studere Candida albicans bindestrek morfogenese i mage-tarmkanalen

Published: July 01, 2020
doi:
10.3791/61488
, ,
1College of Veterinary Medicine,Midwestern University, 2Masters in Biomedical Science Program, College of Graduate Studies,Midwestern University, 3Department of Pathology and Population Medicine, College of Veterinary Medicine,Midwestern University

Summary

Ex vivo analysen beskrevet i denne studien ved hjelp av tarm homogenate ekstrakter og immunofluorescence farging representerer en ny metode for å undersøke bindestrekmorfogenese av Candida albicans i GI-kanalen. Denne metoden kan benyttes til å undersøke miljøsignalene som regulerer morfogenetisk overgang i tarmen.

Abstract

Candida albicans bindestrekmorfogenese i mage-tarmkanalen (GI) er tett kontrollert av ulike miljøsignaler, og spiller en viktig rolle i formidling og patogenese av dette opportunistiske sopppatogenet. Metoder for å visualisere sopphyphae i GI-kanalen in vivo er imidlertid utfordrende, noe som begrenser forståelsen av miljøsignaler for å kontrollere denne morfogeneseprosessen. Protokollen beskrevet her demonstrerer en ny ex vivo metode for visualisering av bindestrekmorfogenese i tarm homogenate ekstrakter. Ved hjelp av en ex vivo analyse viser denne studien at cecal innhold fra antibiotikabehandlede mus, men ikke fra ubehandlede kontrollmus, fremmer C. albicans bindestrekmorfogenese i tarminnholdet. Videre, legge tilbake bestemte grupper av tarm metabolitter til cecal innholdet fra antibiotika-behandlet mus differensialt regulerer bindestrek morfogenese ex vivo. Samlet representerer denne protokollen en ny metode for å identifisere og undersøke miljøsignalene som styrer C. albicans bindestrekmorfogenese i GI-kanalen.

Introduction

Candida albicans er en opportunistisk, polymorf sopppatogen som normalt er commensal, men kan gjennomgå en morfologisk endring i en virulent form som er i stand til å forårsake livstruende infeksjoner hos immunkompromitterte individer1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans er en ledende årsak til systemiske nosocomial infeksjoner, med en 40 \\ u201260% dødelighet selv med antifungal behandling2,14,15. Selv om C. albicans bor i forskjellige vertsnisjemer, inkludert det kvinnelige reproduktivesystemet 16,17,munnhulen til friskeindivider 18 og mage-tarmkanalen (GI)19,20, stammer flertallet av de systemiske infeksjonene fra GI-kanalen og videre, kilden til systemisk infeksjon er ofte bekreftet å være GI tract21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. albicans patogenitet i GI-kanalen påvirkes av et bredt spekter av faktorer; Men en viktig egenskap som er nødvendig for virulens er overgangen fra en gjær celle morfologi til en virulent bindestrekcelle morfologi35,36,37,38,39,40,41,42,43,44. C. albicans vedlegg og spredning fra GI-kanalen under infeksjon er svært forbundet med sin evne til å gå fra en commensal gjær til virulent hyphae, slik at soppene kan forårsake invasiv sykdom44,45,46,47,48,49,50,51,52,53.

En rekke faktorer i tarmen, inkludert n-acetylglucosamine, regulerer bindestrekdannelse av C. albicans. Derfor er det avgjørende å begrense kunnskapsgapet om bindestrekmorfogenesen til dette sopppatogenet iGI-kanalen 54,55,56. Nyere bevis indikerer at ulike tarmmetabolitter differensialt kontrollerer bindestrekmorfogenesen til C. albicans in vitro57,58,59,60. Tekniske begrensninger gir imidlertid problemer når man forsøker å studere C. albicans-hyfaedannelse i in vivo-tarmprøver, spesielt farging av gjær- og bindestrekceller og kvantitativ analyse av bindestrekutvikling. For å forstå C. albicans bindestrekmorfogenese i GI-kanalen, ble en ex vivo-metode utviklet ved hjelp av løselige ekstrakter av homogenisert tarminnhold fra mus for å studere effekten av metabolitter på soppgefokal morfogenese. Ved hjelp av tarmprøver fra mus som er resistente og utsatt for C. albicans GI-infeksjon, vil denne metoden bidra til å identifisere og studere effekten av metabolitter, antibiotika og xenobiotika på sopp bindestrekmorfogenese i GI-kanalen.

Protocol

Alle dyreprotokoller ble godkjent av Midwestern University Institutional Animal Care and Use Committee (IACUC) som beskrevet før57. Institutional Animal Care and Use Committee ved Midwestern University godkjente denne studien under MWU IACUC Protocol #2894. MWU-retningslinjene for dyrepleie følger Folkehelsetjenestens (PHS) retningslinjer for human omsorg og bruk av laboratoriedyr og retningslinjene i dyrevelferdsloven (AWA). 1. Mus studie standard protokoll <li…

Representative Results

Disse resultatene sammen med tidligere funn fra Thangamani-laboratoriet60 indikerer at når C. albicans dyrkes ex vivo i tarmhomogenate ekstrakter tatt fra magen, tynntarmen og tykktarmen av ubehandlet kontroll og antibiotikabehandlede mus, utvikler C. albicans vanligvis med gjærmorfologi (figur 1B). Men når dyrket i cecal ekstrakt fra antibiotikabehandlede mus, C. albicans gjennomgår lett morfogenese, noe som resulterer i prøver som inn…

Discussion

Metoden som er beskrevet her presenterer en ny måte å undersøke effekten av antibiotika, kosttilskudd, xenobiotiske og terapeutiske virkninger på C. albicans bindestrekmorfogenese i GI-kanalen. Siden flertallet av systemiske infeksjoner stammer fra GI tract21,22,23,24,25,26,27<s…

Declarações

The authors have nothing to disclose.

Acknowledgements

Forfatterne anerkjenner ressurser og støtte fra Midwestern University Cellular and Molecular Core Research anlegget.

Materials

1 – 10 µL Pipet Tips Fisher Scientific 02-707-454 Misc
100 – 1000 µL Pipet Tips Fisher Scientific 02-707-400 Misc
20 – 200 µL Pipet Tips Fisher Scientific 02-707-451 Misc
2-methylbutyric acid Sigma 193070-25G hyphal-inhibitory compound
488 goat anti-rabbit IgG Invitrogen (Fisher) A11008 IF Staining secondary ab
Agar Fisher BP1423-500 YPD agar component
Automated Imaging Microscope Keyence BZX700
Candida Albicans Antibody Invitrogen (Fisher) PA1-27158 IF Staining primary ab
cefoperazone Cayman 16113 antibiotic
deoxycholic acid Sigma 30960 hyphal-inhibitory compound
D-Glucose Fisher D16-500 hyphal-promoting compound
forceps Fisher 08-885
lactic acid Alfa Aesar AAAL13242-06 hyphal-inhibitory compound
lithocholic acid Sigma L6250-10G hyphal-inhibitory compound
palmitic acid Sigma P5585-10G hyphal-inhibitory compound
Paraformaldehyde Alfa Aesar A11313 IF Staining fixative
Phosphate-buffered saline (PBS), 10x Alfa Aesar J62692 PBS component
p-tolylacetic acid SCBT sc-257959 hyphal-inhibitory compound
sebacic acid Sigma 283258-250G hyphal-inhibitory compound
sharp ended scissors Fisher 28301
sterile Milli-Q water N/A N/A Misc
YPD Broth BD Biosciences 242810 YPD agar component
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Tags

Ex Vivo AssayCandida AlbicansHyphal MorphogenesisGastrointestinal TractGut MetabolitesEnteric PathogensDissectionGI Tract ExtractionGut Content CollectionC. Albicans CulturePBS ResuspensionGut Content HomogenizationMicroscopy Analysis
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Monasky, R., Villa, S., Thangamani, S. An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract. J. Vis. Exp. (161), e61488, doi:10.3791/61488 (2020).
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